Importance of tRNA interactions with 23S rRNA for peptide bond formation on the ribosome: studies with substrate analogs

Beringer, Malte; Rodnina, Marina V.

doi:10.1515/bc.2007.077

Cited by 21 publications

(29 citation statements)

References 32 publications

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“…The effect of two protons or one proton was observed for C-puromycin at 37°C (30) or 20°C (31), respectively. Furthermore, the rate of peptidyl transfer to CC-puromycin displayed a qualitatively different and much weaker pH-dependence (30).…”

Section: Discussionmentioning

confidence: 96%

“…In fact, the pH-dependence of the peptidyl transfer reaction has previously been observed only for small aa-tRNAanalogs, like puromycin, C-puromycin, CC-puromycin (23,30,31), or puromycin analogs (32). For these, the pH-dependence is complex, with the effect of two protons on the rate of peptidyl transfer to puromycin at 37°C (23,30) as well as at 20°C (31). The effect of two protons or one proton was observed for C-puromycin at 37°C (30) or 20°C (31), respectively.…”

Section: Discussionmentioning

confidence: 99%

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pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA

Johansson

Ieong

Trobro

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

123

164

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We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNA fMet to the aa-tRNAs Phe-tRNA Phe , AlatRNA Ala , Gly-tRNA Gly , Pro-tRNA Pro , Asn-tRNA Asn , and Ile-tRNA Ile , selected to cover a large range of intrinsic pK a -values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, pK obs a , at which the rate was half maximal. The pK obs avalues were downshifted relative to the intrinsic pK a -value of aa-tRNAs in bulk solution. Gly-tRNA Gly had the smallest downshift, while Ile-tRNA Ile and Ala-tRNA Ala had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pK a -values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.ribosome | kinetics | rate limiting step | accommodation | molecular dynamics T he ribosome promotes protein elongation by transfer of the nascent peptide chain from P-site peptidyl-tRNA to A-site aminoacyl-tRNA (aa-tRNA) ( Fig. 1) and translocation of messenger RNA (mRNA) and tRNAs. Peptide bond formation is initiated by a nucleophilic attack of the α-amino group of the amino acid, ester linked to the A-site tRNA, on the ester carbonyl carbon of the peptide chain linked to the 3′-oxygen of the P-site tRNA (Fig. 2). Biochemical data (1-3), crystallographic data (4-6), and molecular dynamics simulations (7,8) have shown that the 2′OH group of A76 of the P-site tRNA greatly accelerates peptide bond formation by providing a shuttle of the proton from the attacking α-amino group to the leaving 3′O of the deacylated P-site tRNA. The rate of ribosomal peptidyl transfer is further accelerated by a network of H-bonds involving water molecules and conserved bases in the peptidyl transferase center (PTC) of the ribosome (4,8,9).Peptidyl transfer requires that the α-amino group of the amino acid on the A-site tRNA is in charge neutral rather than in protonated NH þ 3 form (Fig. 2) (10). At 25°C the pK a -values of the α-amino groups of amino acids in bulk water range from 8.8 (Asn) to 10.6 (Pro) units (11). The pK a -values of the aa-tRNAs, approximated by the pK a -values of the corresponding amino acid methyl and ethyl esters (12)(13)(14), are downshifted in relation to those of the amino acids by two units, with 20°C values ranging from 6.8 (Asn) to 8.6 (Pro) ( Table 1). The rate of ribosomal peptidyl transfer might therefore be expected to vary differently with pH in the physiological range 6-8 for different A-site bound aa-tRNAs. The sensitivity of peptide bond formation to pH-variation in the 6-8 range has so far only been observed for aa-tRNA analogues but not for native aa-tRNAs (15). It has been suggested that the lack of pH sensitivity for aa-tRNAs is that their accommodation in the A site is so slow that the expected p...

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA

Johansson

Ieong

Trobro

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

123

164

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“…Any effect EF-P may have on translocation or on the transition of the ribosome-bound tRNAs to the hybrid states would not be seen by monitoring puromycin reactivity. Furthermore, puromycin does not contain the critical tRNA residues needed for the ratelimiting accommodation step in the A-site (31). It was previously shown that EF-P also affects the rate of reaction between a P-site tRNA and CA-amino acids, which mimic the 3Ј end of an aminoacylated tRNA, but only in the case of small amino acids (32).…”

Section: Discussionmentioning

confidence: 99%

(R)-β-Lysine-modified Elongation Factor P Functions in Translation Elongation

Bullwinkle

Zou

Rajkovic

et al. 2013

Journal of Biological Chemistry

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Background: Post-translational modification activates bacterial elongation factor P (EF-P) in several Gram-negative bacteria.Results: The addition of ␤-lysine alone is sufficient for activation of EF-P to function in translation. Conclusion: Modified EF-P acts by regulating translation of a subset of mRNAs. Significance: EF-P can post-transcriptionally regulate gene expression by controlling translation elongation.

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“…At low concentrations, binding of C-Pmn to the A site appeared to be rate-limiting (data not shown), precluding its use at an affordable concentration. The reaction rate with the longer model substrate CC-Pmn (puromycin derivative corresponding to the complete aminoacylated CCA terminus linked to O-methyltyrosine) or the full-length substrate aminoacyl-tRNA on the 70 S ribosome is partially or completely limited by accommodation in the A site (10,35,36), which makes it difficult to interpret the results for the following peptidyl transfer reaction.…”

Section: Rate Constants Of Pmn Reaction With Differentmentioning

confidence: 99%

Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

Wohlgemuth

Brenner

Beringer

et al. 2008

Journal of Biological Chemistry

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158

194

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The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s ؊1 , regardless of the tRNA identity. The reactivity decreased in the order Lys ‫؍‬ Arg > Ala > Ser > Phe ‫؍‬ Val > Asp Ͼ Ͼ Pro, with Pro being by far the slowest. However, when Phe-tRNA Phe was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was ϳ7 s ؊1 , which corresponds to the rate of binding of Phe-tRNA Phe to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyltRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.The enzymatic activity of the ribosome is to catalyze peptide bond formation. During the peptidyl transfer reaction, the ␣-amino group of aminoacyl-tRNA bound to the A site of the ribosome attacks the ester bond of peptidyl-tRNA in the P site, which results in peptidyl-tRNA extended by one amino acid in the A site and deacylated tRNA in the P site. The tRNA substrates are aligned in the active center of the ribosome by interactions of their CCA ends with 23 S rRNA bases (1-3). The ribosome lowers the activation entropy of the reaction (4, 5) by orienting the reacting groups precisely relative to each other (2, 3), providing an electrostatic environment that reduces the free energy of forming the transition state, shielding the reaction against bulk water (6, 7), or a combination of these effects (8).The peptidyl transfer reaction is modulated by conformational changes at the active site (3, 8 -10) as well as by the nature of the substrates. Rapid peptide bond formation requires fulllength tRNA in both A and P sites, and the reaction rate is influenced by the length of the tRNA fragments when model substrates are used (8, 10 -14). The reaction rate is also influenced by the nature of the amino acid side chain of the A-site substrate (13,(15)(16)(17), but is independent of the nucleophilicity of the attacking amino group in model substrates (18). Moreover, the length of the peptidyl chain and the nature of the C-terminal amino acid of the peptidyl-tRNA in the P site seem to have an effect (10,12,13,19). Early studies with 50 S ribosomal subunits indicated that efficient peptidyl transfer was observed with 3Ј-terminal RNase T1 fragments of N-acetylArg-tRNA Arg and fMet-tRNA fMet as model P-site substrates and an analog of aminoacyl-tRNA, puromycin (Pmn 4 ; O-methyltyrosine linked to N 6 -dimethyladenosine via an amide bond), as A-site substrate (20). In contrast,...

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Importance of tRNA interactions with 23S rRNA for peptide bond formation on the ribosome: studies with substrate analogs

Cited by 21 publications

References 32 publications

pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA

pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA

(R)-β-Lysine-modified Elongation Factor P Functions in Translation Elongation

Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

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